A method of producing a taxane-type diterpene and method of obtaining cultured cells which produce the taxane-type diterpene at a high rate

Abstract: This invention relates to a method of producing a taxane-type diterpene(s) wherein tissues or cells of a plant which produces taxane-type diterpene(s) is cultured in the presence of at least one selected from the group consisting of jasmonic acids, compounds containing a heavy metal, complex ions containing a heavy metal, heavy metal ions, amines and antiethylene agents, a method of producing a taxane-type diterpene wherein the tissues or the cells of the plant are cultured by controlling the oxygen concentration in a gas phase in a culture vessel to less than the oxygen concentration in the atmosphere from the initial stage of the culture, or by controlling the dissolved oxygen concentration in a fluid medium which is in contact with the tissue or the cell to less than the saturated dissolved oxygen concentration at that temperature from the initial stage of the culture, a method of producing a taxane-type diterpene wherein the tissue or the cell of the plant is cultured in a culture vessel, while oxygenic gas containing 0.03 - 10 % of carbon dioxide is used as aeration gas to be introduced to the vessel, and a method of obtaining highly productive cultured cells for the taxane-type diterpene wherein cultured cells of the plant which produces the taxane-type diterpene are separated into a plurality of layers according to the difference in their specific gravities, and the cells contained in at least one layer are cultured, then highly productive cultured cells for the taxane-type diterpene are selected from among those cultured cells. The present invention allows industrial production of a taxane-type diterpene such as taxol which is useful as a therapeutic agent for cancer.

Description: Technical Field This invention relates to a method of producing a taxane-type diterpene including taxol which is useful as a therapeutic agent for ovarian cancer, mammary cancer, lung cancer and the like, and a method of obtaining cultured cells which produce the taxane-type diterpene at a high rate. Background Art Taxol, which is useful as a therapeutic agent for ovarian cancer, mammary cancer, lung cancer and the like, is a taxane-type diterpene identified after being isolated from Taxus brevifolia NUTT, which is a plant belonging to genus Taxus, family Taxaceae and has a complex ester group which is related to its activity. Taxol can be found in all the parts of the plant body of Taxus brevifolia NUTT, but the bark has been reported to exceed all others in its content of the taxol. At present, taxol is collected from a natural or a cultivated plant body, however, the plant belonging to genus Taxus grows slowly, and it takes more than 10 years to grow to a height of 20 cm above the ground, besides the tree dies after its bark is taken off, thus it has been difficult to easily obtain a large amount of taxol. It would be advantageous if a taxane-type diterpene such as taxol and baccatin III which is a precursor of taxol, can be produced by the use of tissue culture, since a large amount of taxol can be easily obtained without cutting down the trees. As a conventional method of producing taxol by utilizing cultured plant cells, a US patent was issued on a production method utilizing cultured cells of Taxus brevifolia NUTT (USP No. 5,019,504), however, the yield of taxol production described therein is 1 - 3 mg/l, and that is insufficient for the industrial production. Besides, the production of taxol by the cell culture is unstable and even when a primary cell of high productivity can be obtained by selection, it is difficult to keep its content by subculturing [E.R.M.Wickremesine et al., World Congress on Cell and Tissue Culture (1992)]. On the other hand, as a prior art in the taxol production, a semisynthetic method from baccatin III, which is a precursor in biosynthesis of taxol, is disclosed in the specification of USP No. 5,015,744 issued to Holton et al. By the use of the plant tissue culture, a raw material for the semisynthetic process such as baccatin III can be produced, thus the plant tissue culture can be also utilized for taxol production by the above-mentioned semisynthetic process. Disclosure of Invention The first object of the present invention is to provide a simple method of producing a taxane-type diterpene by plant tissue culture. The second object of the present invention is to provide a method of obtaining cultured cells which produce a taxane-type diterpene at a high rate. The invention of the present application is a method of producing a taxane-type diterpene wherein a tissue or a cell of a plant which produces a taxane-type diterpene is cultured by controlling the oxygen concentration in a gas phase in a culture vessel to less than the oxygen concentration in the atmosphere, from the initial stage of the culture, or by controlling the dissolved oxygen concentration in a fluid medium which is in contact with the tissue or the cell to less than the saturated dissolved oxygen concentration at that temperature, from the initial stage of the culture, then the taxane-type diterpene is recovered from the resulting cultures. The present invention will be described in further detail. The taxane-type diterpene, which is an object for the present invention, is not particularly limited to any diterpene as far as it has a taxane skeleton, and the illustrative examples include taxol, 7-epitaxol, baccatin III, 7-epibaccatin III, cephalomannine, 7-epicephalomannine, 10-deacetylbaccatin III, 10-deacetylcephalomannine, 10-deacetyltaxol, taxagifine, an analogue thereof, taxane la, an analogue thereof, xylosyl cephalomannine, xylosyl taxol and the like. Examples of the plant to be used in the present invention which produces the taxane-type diterpene are those belonging to genus Taxus, such as Taxus baccata LINN, Taxus cuspidata SIEB. et ZUCC, Taxus cuspidata SIEB. et ZUCC var. nana REHDER, Taxus brevifolia NUTT, Taxus canadiensis MARSH, Taxus chinensis, and Taxus media. According to the invention of the present application, the culture of the plant means culture of a tissue or a cell of the plant, wherein the culture is carried out by a conventionally known process except that the culture is carried out by controlling the oxygen concentration in the gas phase of the culture vessel to below the atmospheric oxygen concentration from the initial stage of the culture, or by controlling the dissolved oxygen concentration in the fluid medium which is in contact with the tissue or the cell to below the saturated dissolved oxygen concentration at that temperature from the initial stage of the culture. Here, by the initial stage of the culture, we refer to from the time when the culture was started through the 7th day after the start of the culture, and the controlling of the oxygen concentration in the gas phase in the culture vessel or the controlling of the dissolved oxygen concentration in the fluid medium which is in contact with the tissue or the cell is preferably done from the beginning of the culture. The controlling period is not particularly limited, and, the controlling under the said conditions can be done in the entire culture period, or only in a part of the entire culture period, however, it is preferable to carry out the control at least for 3 days during the entire culture period. So far, in the culture of a plant producing the taxane-type diterpene, there has been no reports wherein the culture is carried out under such conditions that the oxygen concentration in the gas phase to be supplied to the culture vessel wherein the tissue or the cells are cultured or the dissolved oxygen concentration in the medium which is in contact with the tissue or the cells to below the atmospheric oxygen concentration or below the saturated dissolved oxygen concentration, and it was beyond all expectations that the amount of the taxane-type diterpene to be produced is increased by that. According to the invention of the present application, the oxygen concentration in the gas phase of the culture vessel wherein the tissue or the cells are cultured is required to be controlled to 4 - 15 %, it is particularly preferably controlled to 6 - 12 %. The dissolved oxygen concentration of the fluid medium which is in contact with the tissue or the cells is required to be controlled to 1 - 75 % of the saturated dissolved oxygen concentration at that temperature, it is particularly preferably controlled to 10 - 75 %. Examples of a medium to be used in the invention of the present application, include the medium conventionally known for the tissue culture of a plant, such as medium of Murashige & Skoog (1962), medium of Linsmaier Skoog (1965), Woody Plant Medium (1981), Gamborg's B-5 medium, and Mitsui's M-9 medium and the like. A phytohormone, and if necessary a carbon source, an inorganic component, vitamins, amino acids and the like may be added as well to these media. As a carbon source, a disaccharide such as sucrose, maltose, and lactose, a monosaccharide such as glucose, fructose and galactose, starch or a mixture of two or more kinds of such sugar sources mixed at an appropriate ratio can be utilized. As an inorganic component, illustrative examples include phosphorus, nitrogen, potassium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, copper, molybdenum, chlorine, sodium, iodine and cobalt, and these components can be added in the form of such a compound as potassium nitrate, sodium nitrate, calcium nitrate, potassium chloride, potassium monohydrogenphosphate, potassium dihydrogenphosphate, calcium chloride, magnesium sulfate, sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, zinc sulfate, boric acid, copper sulfate, sodium molybdate, molybdenum trioxide, potassium iodide, cobalt chloride and the like. As the phytohormone, for example, auxin such as indoleacetic acid (IAA), naphthalenacetic acid (NAA), and 2,4-dichlorophenoxy acetic acid (2,4-D), and cytokinin such as kinetin, zeatin, and dihydrozeatin can be used. As the vitamins, for example, biotin, thiamin (vitamin B 1 ), pyridoxine (vitamin B 6 ), pantothenic acid, inositol, nicotinic acid and the like can be used. As the amino acids, for example, glycine, phenylalanine, leucine, glutamine, cysteine and the like can be added. Generally, the carbon source in a concentration of about 1 - about 30 g/l, the inorganic component in a concentration of about 0.1 µM - about 100 mM, the phytohormones in a concentration of about 0.01 - about 10 µM, and the vitamins and the amino acids respectively in a concentration of about 0.1 - about 100 mg/l are used. According to the invention of the present application, both a liquid medium and such a solid medium that contains agar and gelan gum normally in an amount of 0.1 - 1 % can be used. According to the tissue culture of the invention of the present application, a piece of a tissue or a cell of a root, a growing point, a leaf, a stem, a seed, a pollen, an anther and a calyx and the like of the said plant or cultured cells which are obtained by the tissue culture thereof in the above-mentioned medium or another conventional medium can be used. The invention of the present application can also be applied to neoplastic cell and/or hairy-root, obtained by infection with Agrobacterium tumefaciens or Agrobacterium rhizogenes . When these tissues or cells are cultured by controlling the oxygen concentration in the gas phase in the culture vessel to less than the oxygen concentration in the atmosphere, from the initial stage of the culture, or by controlling the dissolved oxygen concentration in the fluid medium which is in contact with the tissue or the cell to less than the saturated dissolved oxygen concentration at that temperature, from the initial stage of the culture, cultured tissue or the cultured cells having higher taxane-type diterpene productivity than that of those obtained by the tissue culture carried out under normal culture conditions can be obtained. According to the invention of the present application, the initial stage of the culture refers to from the time when the culture was started through the 7th day after the start of the culture, and the controlling of the oxygen concentration in the gas phase in the culture vessel or the controlling of the dissolved oxygen concentration in the fluid medium which is in contact with the tissue or the cell is preferably done from the beginning of the culture. The controlling period is not particularly limited, and, the controlling under the said condition can be done in the entire culture period, or only in a part of the entire culture period, however, it is preferable to carry out the control at least for 3 days during the entire culture period. The production method according to the invention of the present application can be used together with a culture method carried out in the presence of various kinds of taxane-type diterpene production promoting substances to further increase the productivity of the taxane-type diterpene. Examples of the taxane-type diterpene production promoting substance include, for example, jasmonic acids represented by the following general formulae (I), (II) or (III): wherein, R 1a , R 1b , R 1c , R 1d , R 1e and R 1f respectively represent hydrogen atom, hydroxyl group, alkyl group having 1 to 6 carbon atoms, or alkoxy group having 1 to 6 carbon atoms; R 2 , R 3 , R 4 , R 5 and R 6a respectively represent hydrogen atom or alkyl group having 1 to 6'carbon atoms; a side chain consisting of C 1 - C 2 - C 3 - C 4 - C 5 - C 6 may contain one or more double bonds; R 6b represents hydroxyl group or -O- carbohydrate residue; R 7 represents hydroxyl group, OM (wherein M is alkali metal atom, alkaline earth metal atom or NH 4 ), NHR 8 (wherein R 8 represents hydrogen atom, acyl group having 1 to 6 carbon atoms, alkyl group having 1 to 6 carbon atoms or amino acid residue), OR 9 (wherein R 9 is alkyl group having 1 to 6 carbon atoms or carbohydrate residue), or alkyl group having 1 to 6 carbon atoms; n is an integer of 1-7; and in the above-mentioned five-membered ring, a double bond may be formed between the neighboring member carbon atoms, wherein, R 1a , R 1b , R 1c , R 1d , R 1e and R 1f respectively represent hydrogen atom, hydroxyl group, alkyl group having 1 to 6 carbon atoms, or alkoxy group having 1 to 6 carbon atoms; R 2 , R 3 , R 4 , R 5 and R 6 respectively represent hydrogen atom or alkyl group having 1 to 6'carbon atoms; a side chain consisting of C 1 - C 2 - C 3 - C 4 - C 5 - C 6 may contain one or more double bonds; R 7 represents hydroxyl group, OM (wherein M is alkali metal atom, alkaline earth metal atom or NH 4 ), NHR 8 (wherein R 8 represents hydrogen atom, acyl group having 1 to 6 carbon atoms, alkyl group having 1 to 6 carbon atoms or amino acid residue), OR 9 (wherein R 9 is alkyl group having 1 to 6 carbon atoms or carbohydrate residue), or alkyl group having 1 to 6 carbon atoms; n is an integer of 1-7; and in the above-mentioned five-membered ring, a double bond may be formed between the neighboring member carbon atoms, wherein, R 1a , R 1b , R 1c , R 1d , R 1e and R 1f respectively represent hydrogen atom, hydroxyl group, alkyl group having 1 to 6 carbon atoms, or alkoxy group having 1 to 6 carbon atoms; R 2 , R 3 , R 4 , R 5 and R 6 respectively represent hydrogen atom or alkyl group having 1 to 6 carbon atoms; a side chain consisting of C 1 - C 2 - C 3 - C 4 - C 5 - C 6 may contain one or more double bonds; R 7 represents hydroxyl group, OM (wherein M is alkali metal atom, alkaline earth metal atom or NH 4 ), NHR 8 (wherein R 8 represents hydrogen atom, acyl group having 1 to 6 carbon atoms, alkyl group having 1 to 6 carbon atoms or amino acid residue), OR 9 (wherein R 9 is alkyl group having 1 to 6 carbon atoms or carbohydrate residue), or alkyl group having 1 to 6 carbon atoms; n is an integer of 1-7; and in the above-mentioned five-membered ring, a double bond may be formed between the neighboring member carbon atoms. Preferable examples of jasmonic acids represented by the above-mentioned general formula (I) include a compound represented by the general formula (I'): wherein, R 1' represents hydrogen atom or hydroxyl group; a side chain consisting of C 1 - C 2 - C 3 - C 4 - C 5 - C 6 may contain a double bond between C 1 and C 2 , between C 2 and C 3 , or between C 3 and C 4 ; R 6b represents hydroxyl group or -O- carbohydrate residue; R 7' represents hydroxyl group, OM (wherein M is alkali metal atom, alkaline earth metal atom or NH 4 ), NHR 8' (wherein R 8' represents hydrogen atom, acyl group having 1 to 4 carbon atoms, alkyl group having 1 to 4 carbon atoms or amino acid residue) or OR 9' (wherein R 9' represents alkyl group having 1 to 4 carbon atoms or carbohydrate residue); n is an integer of 1-7; and in the above-mentioned five-membered ring, a double bond may be formed between the neighboring member carbon atoms, and preferable examples of jasmonic acids represented by the above-mentioned general formula (II) include a compound represented by the general formula (II'): wherein, R 1' represents hydrogen atom or hydroxyl group; a side chain consisting of C 1 - C 2 - C 3 - C 4 - C 5 - C 6 may contain a double bond between C 1 and C 2 , between C 2 and C 3 , or between C 3 and C 4 ; R 7' represents hydroxyl group, OM (wherein M is alkali metal atom, alkaline earth metal atom or NH 4 ), NHR 8' (wherein R 8' represents hydrogen atom, acyl group having 1 to 4 carbon atoms, alkyl group having 1 to 4 carbon atoms or amino acid residue) or OR 9' (wherein R 9 represents alkyl group having 1 to 4 carbon atoms or carbohydrate residue); n is an integer of 1-7; and in the above-mentioned five-membered ring, a double bond may be formed between the neighboring member carbon atoms, and preferable examples of jasmonic acids represented by the above-mentioned general formula (III) include a compound represented by the general formula (III'), wherein, R 1' represents hydrogen atom or hydroxyl group; a side chain consisting of C 1 - C 2 - C 3 - C 4 - C 5 - C 6 may contain a double bond between C 1 and C 2 , between C 2 and C 3 , or between C 3 and C 4 ; R 7' represents hydroxyl group, OM (wherein M is alkali metal atom, alkaline earth metal atom or NH 4 ), NHR 8' (wherein R 8' represents hydrogen atom, acyl group having 1 to 4 carbon atoms, alkyl group having 1 to 4 carbon atoms or amino acid residue) or OR 9' (wherein R 9' represents alkyl group having 1 to 4 carbon atoms or carbohydrate residue); n is an integer of 1-7; and in the above-mentioned five-membered ring, a double bond may be formed between the neighboring member carbon atoms. In the above-mentioned general formulae (I), (II) and (III), examples of alkyl group having 1 to 6 carbon atoms represented by R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , R 2 , R 3 , R 4 , R 5 , R 6 , R 6a , R 7 , R 8 or R 9 include, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, and n-hexyl groups. In the above-mentioned general formulae (I), (II) and (III), examples of alkoxy group having 1 to 6 carbon atoms represented by R 1a , R 1b , R 1c , R 1d , R 1e or R 1f include, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, t-butoxy, n-pentyloxy and n-hexyloxy groups. When R 7 is OM, examples of an alkali metal atom or an alkaline earth metal atom represented by M include, for example, sodium, potassium and calcium. When R 7 is NHR 8 , the acyl group having 1 to 6 carbon atoms represented by R 8 may have either a straight chain or a branched chain, and their examples include, for example, formyl, acetyl, propionyl, butyryl, valeryl, hexanoyl and acryloyl groups. When R 7 is NHR 8 , examples of an amino acid residue represented by R 8 include isoleucyl, tyrosyl, and tryptophyl groups. When R 7 is OR 9 , an example of a carbohydrate residue represented by R 9 is glucopyranosyl group, and when R 6b is -O- carbohydrate residue in the above-mentioned general formula (I), an example of a carbohydrate residue is glucopyranosyl group. In the compounds represented by the general formulae (I), (II) and (III), a double bond may be formed between the neighboring member carbon atoms in the five-membered ring. Illustrative examples of the compound represented by the general formula (I) include those shown as follows; Illustrative examples of the compound represented by the general formula (II) include those shown as follows; Illustrative examples of the compound represented by the general formula (III) include those shown as follows; (Compound I) R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , R 2 , R 3 , R 4 , R 5 , R 6 : H A double bond is formed between C 3 and C 4 . R 7 : -OH or -OCH 3 n: 1 to 3 (Compound J) R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , R 2 , R 3 , R 4 , R 5 , R 6 : H R 7 : -OH n: 1 Illustrative examples of the compound represented by the general formula (III) wherein R 1a , R 1b , R 1c , R 1d , R 1e or R 1f is hydroxyl group, or a double bond is formed between the neighboring member carbon atoms in the five-membered ring, include those shown as below; (1) n = 1, R = H (2) n = 7, R = H (3) n = 7, R = OH (1) R=H (2) R=OH Preferable examples of a compound represented by the general formula (I), (II) or (III) include the compounds wherein R 1a , R 1b , R 1c , R 1d , R 1c , R 1f , R 2 , R 3 , R 4 , R 5 and R 6 are hydrogen atoms, R 7 is hydroxyl group or methoxy group, and a side chain consisting of C 1 -C 2 - C 3 - C 4 - C 5 - C 6 does not contain a double bond, or contain a double bond between C 1 and C 2 , between C 2 and C 3 , or between C 3 and C 4 . Jasmonic acids to be used in the present invention which are represented by the general formula (I), (II) or (III) have various stereoisomers (cis-trans isomers and optical isomers), and each isomer can be used alone or in the form of a mixture. All of the jasmonic acids shown above have the effect of improving the productivity in the taxane-type diterpene production, however, tuberonic acid, methyl tuberonate, cucurbic acid, methyl cucurbate, jasmonic acid and methyl jasmonate, which are the compounds represented by the general formula (I), (II) or (III) 'wherein R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , R 2 , R 3 , R 4 , R 5 and R 6 are hydrogen atoms, R 7 is hydroxyl group or methoxy group, n is 1, and a double bond is formed between C 3 and C 4 , are particularly preferable from the view point of their high effectiveness in improving the productivity. These jasmonic acids are prepared by synthesis or extraction and the like from a plant (H.Yamane et al. Agric. Biol. Chem., 44 , 2857-2864(1980)). Further examples of the taxane-type diterpene production promoting substance include, for example, compounds containing a heavy metal, complex ions containing a heavy metal, heavy metal ions, amines and antiethylene agents. The heavy metals which may be used as taxane-type diterpene production promoting substance, are not particularly limited to any heavy metal as far as it belongs to the copper group or the iron group, however, as the metal belonging to the copper group, the use of silver is particularly preferable, and as the metal belonging to the iron group, the use of cobalt is particularly preferable. In addition to that, when silver or cobalt is used, it is preferably used in the form of a compound containing the said heavy metal, a complex ion containing the said metal or in the form of the said metal ion. These compounds can be used alone or in combination. Illustrative examples of the compound containing silver include silver nitrate, silver sulfate, silver fluoride, silver chlorate, silver perchlorate, silver acetate, silver sulfite, silver hexafluorophosphate(V), silver tetrafluoroborate, diamine silver(I) sulfate, potassium diaminoargentate(I) and the like. Among these, particularly preferable compounds can be exemplified by silver nitrate, silver sulfate and the like. Illustrative examples of the complex ion containing silver include [Ag(S 2 O 3 ) 2 ] 3- , [Ag(S 2 O 3 ) 3 ] 5- , [Ag(NH 3 ) 2 ] + , [Ag(CN) 2 ] - , [Ag(CN) 3 ] 2- , [Ag(SCN) 2 ] - , [Ag(SCN) 4 ] 3- and the like. Among these, particularly preferable complex ions can be exemplified by [Ag(S 2 O 3 ) 2 ] 3- , [Ag(S 2 O 3 ) 3 ] 5- and the like. Illustrative examples of the compound containing cobalt include cobalt chloride, cobalt nitrate, cobalt sulfate, cobalt fluoride, cobalt perchlorate, cobalt bromide, cobalt iodide, cobalt selenate, cobalt thiocyanate, cobalt acetate, ammonium cobalt sulfate, cobalt(II) potassium sulfate, hexaamminecobalt(III) chloride, pentaammineaquacobalt(III)chloride, nitropentaamminecobalt(III) chloride, dichlorotetraamminecobalt(III) chloride hemihydrate, dinitrotetraamminecobalt(III) chloride, carbonatotetraamminecobalt(III) chloride, ammonium tetranitrodiamminecobaltate(III), sodium hexanitrocobaltate(III), tris(ethylenediamine)cobalt(III) chloride trihydrate, dichlorobis(ethylenediamine)cobalt(III) chloride, potassium tris(oxalato)cobaltate(III) trihydrate, potassium hexacyanocobaltate(III), potassium (ethylenediaminetetraacetato)cobaltate(III) dihydrate, hydridotetracarbonylcobalt(I), dicarbonyl(cyclopentadienyl)cobalt(I), octacarbonyldicobalt(O), hexacarbonyl(acetylene)dicobalt(0), bis(cyclopentadienyl)cobalt(I), (cyclopentadienyl)(1,5-cyclooctadiene)cobalt(I) and the like. Among these, particularly preferable compounds can be exemplified by cobalt chloride, cobalt nitrate, cobalt sulfate and the like. Illustrative examples of the complex ion containing cobalt include pentaammineaquacobalt ion, nitropentaamminecobalt ion, dichlorotetraamminecobalt ion, dinitrotetraamminecobalt ion, carbonatotetraamminecobalt ion, tetranitrodiamminecobalt ion, hexanitrocobalt ion, tris(ethylenediamine)cobalt ion, dichlorobis(ethylenediamine)cobalt ion, tris(oxalato)cobalt ion, hexacyanocobalt ion, (ethylenediaminetetraacetato)cobalt ion and the like. Of the said heavy metals, the compound containing silver, the complex ion containing silver or the silver ion preferably has a concentration in the medium of 10 -8 M - 10 -1 M, and it is further preferable to adjust the concentration to be in the range of 10 -7 M to 10 -2 M. The compound containing cobalt, the complex ion containing cobalt or the cobalt ion preferably has a concentration in the medium of 10 -6 - 10 -1 M, and it is further preferable to adjust the concentration to be in the range of 10 -5 to 10 -2 M. As the amines which may be used as taxane-type diterpene production promoting substance both monoamines and polyamines can be used, however, the use of polyamines is particularly preferable. The term amine is used herein to refer to an amine or a salt thereof. In addition to that, examples of the amines include mono, di or trialkyl amines wherein a part of hydrogen atoms in the alkyl group may be substituted by hydroxyl group, such as methyl amine, ethyl amine, dimethyl amine, diethyl amine, triethyl amine, diethanol amine, triethanol amine or a salt thereof; polymethylene diamine wherein the polymethylene moiety may be interrupted by imino group, and H in the amino group can be substituted by lower alkyl group, such as putrescine, cadaverine, spermidine, spermin, ethylenediamine, N,N-diethyl-1,3-propane diamine, triethylene tetramine, or a salt thereof; cyclic alkyl amine such as cyclopentyl amine, cyclohexyl amine or a salt thereof, or a cyclic amine such as methenamine and piperazine, or a salt thereof. Among these amines, preferable amines can be exemplified by polyamines such as putrescine [NH 2 (CH 2 ) 4 NH 2 ], cadaverine [NH 2 (CH 2 ) 5 NH 2 ], spermidine [NH 2 (CH 2 ) 3 NH(CH 2 ) 4 NH 2 ], spermin [NH 2 (CH 2 ) 3 NH(CH 2 ) 4 NH(CH 2 ) 3 NH 2 ], ethylene diamine [NH 2 (CH 2 ) 2 NH 2 ], N,N-diethyl-1,3-propane diamine [(C 2 H 5 ) 2 N(CH 2 ) 3 NH 2 ], diethylene triamine [NH 2 (CH 2 ) 2 NH(CH 2 ) 2 NH 2 ] and the like or a salt thereof. The said amines preferably have a concentration in the medium of 10 -8 M - 10 -1 M, and it is further preferable to adjust the concentration to be in the range of 10 -7 M to 10 -2 M. The antiethylene agent, which is used as taxane-type diterpene production promoting substance is not particularly limited to any specific substance as far as it is a substance which inhibits the ethylene biosynthesis mechanism of the cultures and/or a substance which removes the ethylene remaining in the cultures or existing in the gas phase or in the medium in the culture vessel containing the cultures. Illustrative examples of a method to inhibit the ethylene biosynthesis mechanism include a method of inhibiting the activity of an enzyme which catalyzes the conversion of S-adenosyl-methionine into 1-aminocyclopropane-1-carboxylic acid, and a method of inhibiting the activity of an enzyme which catalyzes the conversion of 1-aminocyclopropane-1-carboxylic acid into ethylene, and illustrative examples of the compound having the former function include, aminoxyacetic acid, acetylsalicylic acid, Rhizobitoxine, aminoethoxyvinylglycine, methoxyvinylglycine, a-aminoisobutyric acid, 2,4-dinitrophenol and the like. They can also include a salt, an ester, an amino acid derivative and a carbohydrate derivative of the said compound. Illustrative examples of the salt include sodium, potassium, calcium, and magnesium salts, illustrative examples of the ester include methyl, ethyl, propyl, and butyl esters, illustrative examples of the amino acid derivatives include glycine, methionine, and phenylalanine derivatives, and illustrative examples of the carbohydrate derivative include glucose and maltose derivatives. The salt, ester, amino acid derivative, carbohydrate derivative according to the present invention are not limited to the above-mentioned compounds. Illustrative examples of the compound having the latter function include gallic acid, a salt, an ester, an amino acid derivative and a carbohydrate derivative thereof [Hiroshi Hyodo, Society of Horticulture Autumn Convention 1987 Symposium Summary, p. 122, Susumu Kuraishi, Phytohormone, Tokyo University Publication, p.111]. Illustrative examples of the salt include sodium, potassium, calcium, and magnesium salts, illustrative examples of the ester include methyl, ethyl, propyl, and butyl esters, illustrative examples of the amino acid derivatives include glycine, methionine, and phenylalanine derivatives, and illustrative examples of the carbohydrate derivatives include glucose and maltose derivatives. The salt, ester, amino acid derivative, carbohydrate derivative according to the present invention are not limited to the above-mentioned compounds. Illustrative examples of the substance which removes the ethylene remaining in the cultures or existing in the gas phase or the medium in the culture vessel containing the cultures include 1,5-cyclooctadiene and isothiocyanic acid, a salt, an ester (such as allyl isothiocyanate and benzyl isothiocyanate), an amino acid derivative and a carbohydrate derivative thereof [Megumi Munakata, Chemical control in plants, 29 (1), 89-93 (1994)]. Illustrative examples of the salt include sodium, potassium, calcium, and magnesium salts, illustrative examples of the ester include methyl, ethyl, propyl, butyl, and allyl esters, illustrative examples of the amino acid derivatives include glycine, methionine, and phenylalanine derivatives, and illustrative examples of the carbohydrate derivatives include glucose and maltose derivatives. The salt, ester, amino acid derivative, carbohydrate derivative according to the present invention are not limited to the above-mentioned compounds. The antiethylene agent is required to have a concentration in a culture medium of 10 -8 M - 10 -1 M, and it is particularly preferable to control the concentration of the antiethylene agent to be in the range of 10 -7 M to 10 -2 M. Also the invention of the present application can be also used together with a method wherein the cells are fractionated into a plurality of layers according to the difference in their specific gravities, and the cells contained in at least one layer are cultured. The production method according to the invention of the present application can be used together with both the said method wherein culture is carried out in the presence of the jasmonic acids and the like and the method wherein the cells are fractionated into a plurality of layers according to the difference in their specific gravities and cells contained in at least one layer are cultured. Taxane-type diterpene can be fractionated from the cultures such as cultured tissues, cultured cells and culture medium, which are obtained according to the above-mentioned process, by extraction with an organic solvent such as methanol. One preferable example of the tissue culture according to the invention of the present application can be illustrated as follows. A piece of a plant body of a plant belonging to genus Taxus, such as a root, a growing point, a leaf, a stem, a seed and the like is sterilized and placed on Woody Plant Medium solidified with gelan gum, and kept at 10 - 35 °C for about 14 - 60 days so that a part of the tissue piece is changed to callus. By subculturing the callus thus obtained, the growing speed is gradually increased and stabilized callus can be obtained. By the stabilized callus, we refer to a callus which remains in callus state during culture without showing differentiation into a shoot or a root and the cells of which have uniform growing speed. Such stabilized callus is transferred to a liquid medium, suited for the growth, such as liquid Woody Plant Medium and grown. The growing speed is further increased in the liquid medium. According to the present invention, the stabilized callus or the cells constituting the above-mentioned callus is grown under the culture conditions wherein the oxygen concentration in a gas phase in a culture vessel is controlled to less than the oxygen concentration in the atmosphere from the initial stage of the culture, or the dissolved oxygen concentration in a fluid medium which is in contact with the tissue or the cell is controlled to less than the saturated dissolved oxygen concentration at that temperature, from the initial stage of the culture. The tissue or the cell gains energy necessary for maintenance and growth of individium, by consuming oxygen (respiration). It is generally known that when a tissue or a cell is cultured, the cell mass is increased and the amount of the oxygen consumption is increased as well with the passage of culture period. Accordingly unless a ventilation gas is forcedly supplied from outside of the system, the oxygen concentration in the gas phase in the culture vessel such as a flask wherein the tissue or the cell is contained, or the dissolved oxygen concentration in the medium which is in contact with the tissue or the cell naturally decreases to a value less than the oxygen concentration in the atmosphere, or the saturated dissolved oxygen concentration at that temperature, with the passage of culture period. The present invention is different from the above-mentioned finding on the point that the culture is carried out by actively controlling the oxygen concentration in the gas phase in the culture vessel which contains the tissue or the cell or the dissolved oxygen concentration in the culture medium to less than the oxygen concentration in the atmosphere or the saturated dissolved oxygen concentration at that temperature. In one illustrative process to enhance the effect of the present invention, the oxygen concentration in the gas phase in the culture vessel or the dissolved oxygen concentration in the fluid medium is previously controlled to less than the oxygen concentration in the atmosphere or the saturated dissolved oxygen concentration at that temperature, prior to the subculture of the tissue or the cell in the culture vessel. The controlling period is not particularly limited as mentioned above, however, it is preferable to carry out the control at least for 3 days during the entire culture period. In addition to that, the controlling method is not particularly limited to any as far as it is a method wherein the oxygen concentration in the gas phase in the culture vessel which contains the tissue or the cell, or the dissolved oxygen concentration in the fluid medium which is in contact with the tissue or the cell, can be controlled to less than the oxygen concentration in the atmosphere or the saturated dissolved oxygen concentration at that temperature, and in some examples of such method, a gas having a controlled oxygen concentration, which is obtained by mixing an air with nitrogen and the like to lower the oxygen concentration, is directly sent into the gas phase in the culture vessel or the culture medium, or such a gas is directly sent into the culture medium outside of the culture vessel, i.e. in an aeration tank and the like, then the culture medium is poured into the culture vessel, or a gas such as air to be supplied to the culture vessel is directly sent into the gas phase or the culture medium by controlling the feed speed, or such a gas is directly sent into the culture medium outside of the culture vessel, i.e. in an aeration tank and the like then the culture medium is poured into the culture vessel, or the culture vessel is placed under low oxygen atmosphere to carry out culture or the culture is carried out in the presence of an oxygen adsorbing agent. The temperature for the tissue culture according to the present invention is usually about 10 - about 35 °C, and preferably about 23 - 28 °C according to the high growing speed. As for the culture period, 14 - 42 days are preferable. When a liquid medium is used for the culture according to the present invention, the cultured cells can be fractionated from the culture medium after the culture is completed by such a method as decantation or filtration and the desired taxane-type diterpene can be fractionated from the cultured cells and/or the culture medium by such a method as extraction with an organic solvent. It is also possible to recover the desired compound continuously during the culture by allowing an adsorbing agent or an appropriate organic solvent coexist in the culture system. According to the invention of the present application, taxane-type diterpene can be easily obtained in large quantity. When the invention of the present application is to be industrially executed, the efficiency can be further increased by employing the following further inventions of the present application in an independent form or in a combined form. That means, it is necessary to supply a gas containing oxygen to a culture liquid to culture tissues or cells of a plant which produces taxane-type diterpene. Normally, air is used for this purpose, however, after an intensive study, the present inventors found that the taxane-type diterpene production can be efficiently carried out by the use of a gas containing 0.03 - 10 %, preferably 0.1 - 5 % of carbon dioxide, as a gas to be introduced to a tank for culturing the tissues or the cells of the plant producing the taxane-type diterpene, and completed the first further invention of the present application. The present inventors also found that the productivity of the taxane-type diterpene in the cultures can be remarkably improved and the fluctuation of the taxane-type diterpene productivity due to the subculture can be controlled by carrying out a two-stage culture of the tissue or the cell of the plant producing the taxane-type diterpene, comprising a first stage using a medium to which an oxidizing agent or a water soluble organic compound containing oxygen is added for obtaining the tissues or the cells which is activated for production of the taxane-type diterpene in the subsequent stage, and a second stage which is carried out such conditions that promote the production of the taxane-type diterpene, and completed the second further invention of the present application. Here, examples of the oxidizing agent include peroxodisulfates such as potassium peroxodisulfate and hydrogen peroxide, and examples of the water soluble organic compound containing oxygen include dimethyl formamide, dimethyl sulfoxide, and ethylene glycol and the like. The total concentration of the above-mentioned additive in the culture medium is preferably 10 -6 M - 10 -1 M immediately after the addition, and it is further preferable to control the concentration to be in the range of 10 -5 M to 10 -2 M. The present inventors also found that the high density culture of the tissue or the cell of the plant producing the taxane-type diterpene can be carried out by inoculating the tissues or the cells in a culture medium containing a saccharide in a concentration of 2 - 50 g/l, preferably 10 - 30 g/l, and/or nitrate ion in a concentration of 2 - 50 mmol/l, preferably 10 - 30 mmol/l, then by adding a nutrient source solution containing the saccharide in an amount of 0.2 - 5 g/l, preferably 0.5 - 3 g/l, and/or nitrate ion in an amount of 0.2 - 5 mmol/l, preferably 0.5 - 3 mmol/l per day with respect to the initial volume of the said culture medium, continuously or intermittently to the culture medium, thereby the taxane-type diterpene production volume per culture vessel can be remarkably increased and completed the third further invention of the present application. Here, by the density, we refer to a cell mass per volume of the culture solution in the culture vessel, which is shown in terms of dry cell mass (g) per liter of the culture solution. According to the third further invention of the present application, it is preferable to carry out culture while the culture medium is.renewed by adding the nutrient source solution and simultaneously separating and removing the same volume of the medium from the tissues or the cells and to recover the taxane-type diterpene from at least one selected from the resulting cultures, the medium recovered by removal during the culture, and the medium obtained at the end of the culture. The third further invention of the present application is particularly effective in improving the taxane-type diterpene productivity in the high density culture wherein the density of the tissue or the cell of the above-mentioned plant at the start of the culture with respect to the medium volume is 50 g fresh weight/l or higher. Furthermore, though the culture is normally finished when the cells of high density are obtained, the present inventors achieved, through the intensive study, the continuous culture by continuing the culture while the cells are removed, and after further examination, finally completed a continuous culture method, which is the seventh invention of the present application. That means, the taxane-type diterpene can be produced with such a high rate that could be hardly attained with the conventional process, by adding the fresh medium continuously or intermittently in such a way that the specific renewing ratio defined by the dimensionless number F = V I /V/µ (wherein, V is the total volume of the culture medium in a culture tank, V I is the feed speed of the fresh medium, and µ is the specific growth rate of the tissues or the cells) is in the range of 0.1 to 10, and by recovering the taxane-type diterpene from the culture medium containing the tissues or the cells which is continuously or intermittently taken out from the tank and/or the culture solution which does not contain the tissue nor the cell and which is continuously or intermittently taken out from the tank, and completed the fourth further invention of the present application. It is further preferable to set the specific renewing ratio of the culture medium, F, to 0.5 - 5. The saccharide concentration in the culture solution is preferably 5 - 40 g/l, and the nitrate ion concentration in the culture solution is preferably 10 - 40 mmol/l. The present invention can be effective with the cell density in terms of fresh cell weight per litter of 50 - 500 g, however, the higher the density is as far as it is in a range wherein extremely vigorous stirring is not required, the more efficiently the taxane-type diterpene can be produced, thus the preferable density is 200 g or higher per liter. Brief Description of Drawings Fig. 1 is a diagram illustrating an example of a culture apparatus used for carrying out the tissue culture according to the invention of the present application. Each numeral used in the Fig. 1 has the following meaning. 1 Air feed pipe 2 Nitrogen feed pipe 3 Culture vessel 4 Sparger for supplying oxygenic gas 5 Electrode for dissolved oxygen 6 Dissolved oxygen concentration controller 7 Vent 8 Valve 9 Oxygen flow control valve 10 Air filter 11 Impeller Fig. 2 is a diagram illustrating an example of a culture apparatus used for carrying out the tissue culture according to the third or the fourth further invention of the present application. Each numeral and letter used in the Fig. 2 has the following meaning. 12 Medium feed pipe 13 Medium feed opening 14 Opening with a filter for taking out culture medium alone (the culture medium containing no tissues nor cells) 15 Culture medium outlet pipe 16 Sparger for supplying oxygenic gas 17 Impeller 18 Culture mixture (the culture solution containing tissues or cells) discharge pipe 19 Pressurized fluid inlet a, b, c, d and e Valves Best Mode for Carrying Out the Invention The invention will be further illustrated with the following examples and comparative examples, however, these examples are not to be construed to limit the scope of the invention. [Example 1] A part of stem of Taxus baccata LINN which had been previously sterilized with 2 % antiformin solution or 70 % ethanol solution and the like, was placed on solid Woody Plant Medium (containing gelan gum of 0.25 % by weight) to which naphthalenacetic acid had been added to give the concentration of 10 -5 M, and static culture was carried out at 25 °C in a dark place to provide Taxus baccata LINN callus. One gram (fresh weight) of the callus was inoculated to an Erlenmeyer flask containing 20 ml of liquid Woody Plant Medium to which the above-mentioned component had been added to give the same concentration and shake culture was carried out with a rotary shaker (amplification of 25 mm, 100 rpm) and the callus was subcultured in every 21 days to accelerate the growth rate of the callus. One gram (fresh weight) of the cultured cells thus obtained was inoculated to an Erlenmeyer flask containing 20 ml of liquid Woody Plant Medium to which the above-mentioned component had been added to give the same concentration, and the flask was placed in a chamber (the capacity of 3000 ml) having a gas feed opening and a gas discharge opening, then the chamber was closed hermetically, and air was so mixed with nitrogen that the concentration of the oxygen in a gas to be supplied to the cells to be cultured became 4 - 15 %, and while the gas was supplied through the feed opening at the rate of 25 ml per minute, shake culture was carried out at 25 °C for 21 days. After completing the culture, cultured cells of Taxus baccata LINN were harvested by filtration and lyophilized, then the dry weight was measured to obtain the growth rate thereof. Taxane-type diterpenes were extracted from the dried cells with methanol and the like, and they were determined by comparing with standard taxol, cephalomannine, and baccatin III using high performance liquid chromatography to measure the yields of the taxane-type diterpenes. The results are shown in Table 1. [Comparative Example 1] The procedure of Example 1 was carried out except that the concentration of the oxygen in a gas to be supplied to the cells to be cultured was controlled to be 20 %. The results are shown in Table 1. [Reference Example 1] The procedure of Example 1 was carried out except that the cultured cells inoculated to the flask were cultured in the atmosphere. The results are shown in Table 1. [Example 2] The procedure of Example 1 was carried out except that the concentration of the oxygen in a gas to be supplied to the cells to be cultured was controlled to be 10 % and the mixed gas was supplied from the start of the culture for 3 days, then air was supplied till the end of the culture (for 18 days). The results are shown in Table 1. [Example 3] The procedure of Example 1 was carried out except that the concentration of the oxygen in a gas to be supplied to the cells to be cultured was controlled to be 10 % and the mixed gas was supplied from the start of the culture for 7 days, then air was supplied till the end of the culture (for 14 days). The results are shown in Table 1. [Example 4] The procedure of Example 1 was carried out except that the concentration of the oxygen in a gas to be supplied to the cells to be cultured was controlled to be 10 %, and the mixed gas was supplied from the start of the culture for 14 days, then air was supplied till the end of the culture (for 7 days). The results are shown in Table 1. [Example 5] The procedure of Example 1 was carried out except that methyl ester of jasmonic acid (which is a compound represented by the general formula (III) wherein R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , R 2 , R 3 , R 4 , R 5 , and R 6 are hydrogen atoms, R 7 is methoxy group, n is 1 and C 3 and C 4 have a double bond between them; 90 % of which is in the trans-form, and 10 % of which is in the cis-form) was added as one of jasmonic acids on the 14th day after starting the culture to give the final concentration of 10 - 1000 µM. The results are shown in Table 2. The productivity of taxane-type diterpene could be remarkably improved by combination of supply of oxygen of low concentration and addition of the methyl ester of jasmonic acid. [Example 6] Eighty-five gram (fresh weight) of cultured cells obtained in Example 1 with accelerated growth rate were inoculated in a tank for stirred tank culture (capacity of 3000 ml) having an electrode for a dissolved oxygen concentration and a dissolved oxygen concentration controller, into which 1700 ml of liquid Woody Plant Medium had been poured. Then stirred tank culture was carried out at 25 °C for 21 days while the dissolved oxygen concentration in the medium was controlled to be 0.1 ppm or less by adjusting the mixing ratio of air and nitrogen. The schematic diagram of the culture apparatus is shown in Fig. 1 and the results are shown in Table 3. [Example 7] The procedure of Example 6 was carried out except that the concentration of the dissolved oxygen was controlled to be 1 ppm or less by adjusting the mixing ratio. The results are shown in Table 3. [Example 8] The procedure of Example 6 was carried out except that the concentration of the dissolved oxygen was controlled to be 2 ppm or less by adjusting the mixing ratio. The results are shown in Table 3. [Example 9]. The procedure of Example 6 was carried out except that the concentration of the dissolved oxygen was controlled to be 4 ppm or less by adjusting the mixing ratio. The results are shown in Table 3. [Example 10] The procedure of Example 6 was carried out except that the concentration of the dissolved oxygen was controlled to be 6 ppm or less by adjusting the mixing ratio. The results are shown in Table 3. [Comparative Example 2] The procedure of Example 6 was carried out except that air was supplied. The results are shown in Table 3. [Example 11] The procedure of Example 6 was carried out except that the concentration of the dissolved oxygen in the medium was controlled to be 4 ppm or less from the start of the culture for 3 days by adjusting the mixing ratio, and air was supplied till the end of the culture (for 18 days). The results are shown in Table 3. [Example 12] The procedure of Example 6 was carried out except that the concentration of the dissolved oxygen in the medium was controlled to be 4 ppm or less from the start of the culture for 7 days by adjusting the mixing ratio, and air was supplied till the end of the culture (for 14 days). The results are shown in Table 3. [Example 13] The procedure of Example 6 was carried out, except that the concentration of the dissolved oxygen in the medium was controlled to be 4 ppm or less from the start of the culture for 14 days by adjusting the mixing ratio, and air was supplied till the end of the culture (for 7 days). The results are shown in Table 3. max oxygen concentration in the gas phase (%) growth rate (times) yield of baccatin III (mg/l) yield of cephalomannine (mg/l) yield of taxol (mg/l) total yield of taxane (mg/l) Comparative 20 3.01 0.78 1.71 1.46 3.95 Example 1 Example 1 4 2.09 1.65 5.24 1.78 8.67 " 7 2.60 3.37 7.25 2.49 13.11 " 10 2.90 3.19 7.51 2.96 13.66 " 15 2.82 3.18 6.86 2.18 12.22 Example 2 10 2.79 1.33 4.88 1.74 7.95 Example 3 10 2.62 3.21 7.16 1.75 12.12 Example 4 10 2.66 3.17 7.16 1.92 12.25 Reference Example 1 20 3.03 0.77 1.23 0.95 2.95 max oxygen concentration in gas phase (%) concentration of methyl jasmonate (µM) growth rate (times) yield of baccatin III (mg/l) yield of taxol (mg/l) yield of cephalomannine (mg/l) total yield of taxane (mg/l) Comparative 20 0 3.01 0.78 1.71 1.46 3.95 Example 1 Example 1 10 0 2.90 3.19 7.51 2.96 13.66 Example 5 10 10 2.82 2.26 10.24 1.73 14.73 " 10 100 2.45 11.32 22.44 2.29 36.05 " 10 1000 1.47 2.10 5.04 1.47 8.61 max dissolved oxygen concentration in the medium (ppm) growth rate (times) yield of baccatin III (mg/l) yield of taxol (mg/l) yield of cephalomannine (mg/l) total yield of taxane (mg/l) Comparative 8 (100 ) 3.01 0.58 1.74 0.91 3.23 Example 2 Example 6 0.1 (1.25 ) 2.20 0.70 2.05 1.12 3.87 Example 7 1 (12.5 ) 2.25 1.05 3.69 1.22 5.96 Example 8 2 (25 ) 2.49 2.44 5.54 2.70 10.68 Example 9 4 (50 ) 2.75 3.91 8.14 1.80 13.85 Example 10 6 (75 ) 2.98 2.52 5.69 1.23 9.44 Example 11 4 (50 ) 2.89 1.62 4.02 1.54 7.18 Example 12 4 (50 ) 2.62 3.32 8.46 3.08 14.86 Example 13 4 (50 ) 2.75 3.19 7.72 2.65 13.56 Industrial Applicability The present invention allows industrial production of a taxane-type diterpene including taxol which is useful as a therapeutic agent for ovarian cancer, mammary cancer, lung cancer and the like.

Claims: A method of producing a taxane-type diterpene wherein tissues or cells of a plant which produce a taxane-type diterpene are cultured by controlling the oxygen concentration in a gas phase in a culture vessel to less than the oxygen concentration in the atmosphere from the initial stage of the culture, or by controlling the dissolved oxygen concentration in a fluid medium which is in contact with the tissues or the cells to less than the saturated dissolved oxygen concentration at that temperature from the initial stage of the culture, then the taxane-type diterpene is recovered from the resulting cultures. The method according to claim 1, wherein the tissues or the cells of the plant which produce the taxane-type diterpene are cultured by controlling the oxygen concentration in the gas phase. in the culture vessel to 4 - 15 %, or by controlling the dissolved oxygen concentration in the fluid medium which is in contact with the tissues or the cells, to 1 - 75 % of the saturated dissolved oxygen concentration at that temperature. The method according to claim 1, wherein the tissues or the cells of the plant which produce the taxane-type diterpene are cultured by controlling the oxygen concentration in the gas phase in the culture vessel to 6-12 %, or by controlling the dissolved oxygen concentration in the fluid medium which is in contact with the tissue or the cell, to 10 - 75 % of the saturated dissolved oxygen concentration at that temperature. The method according to claim 1, wherein the tissue or the cell of the plant which produce the taxane-type diterpene is cultured by controlling the oxygen concentration in the gas phase in the culture vessel, or the dissolved oxygen concentration in the fluid medium by adjusting the oxygen concentration in aeration gas to be supplied to the culture vessel and/or to the culture medium, or by adjusting the feed speed of the aeration gas to be supplied to the culture vessel and/or to the culture medium. The method according to claim 1, wherein the controlling of the oxygen concentration in the gas phase in the culture vessel or the controlling of the dissolved oxygen concentration in the fluid medium which is in contact with the tissues or the cells is started between the beginning of the culture and the 7th day after the start of the culture and the controlling is kept at least for 3 days. The method according to claim 1, wherein the taxane-type diterpene is at least one compound selected from the group consisting of taxol, 7-epitaxol, baccatin III, 7-epibaccatin III, cephalomannine, 7-epicephalomannine, 10-deacetylbaccatin III, 10-deacetylcephalomannine, 10-deacetyltaxol, taxagifine, xylosyl cephalomannine, and xylosyl taxol. The method according to claim 1, wherein the plant which produces the taxane-type diterpene is a plant belonging to genus Taxus. The method according to claim 1, wherein the culture is carried out in the presence of jasmonic acids. A method of producing a taxane-type diterpene wherein tissues or cells of a plant which produce a taxane-type diterpene are cultured by carrying out a two-stage culture, comprising a first stage using a medium to which an oxidizing agent or a water soluble organic compound containing oxygen is added and a second stage which is carried out according to the production method of claim 1, then the taxane-type diterpene is recovered from the resulting cultures. The method according to claim 1, wherein the tissues or the cells of the plant which produce the taxane-type diterpene are cultured by inoculating the tissues or the cells in a culture medium containing a saccharide in a concentration of 2 - 50 g/l, and/or nitrate ion in a concentration of 2 - 50 mmol/l, then by adding a nutrient source solution containing the saccharide in an amount of 0.2 - 5 g/l, and/or nitrate ion in an amount of 0.2 - 5 mmol/l per day based on the initial volume of the said culture medium, continuously or intermittently to the culture medium, then the taxane-type diterpene is recovered from the resulting cultures. The method according to claim 10, wherein the culture is carried out while the culture medium is renewed by adding the nutrient source solution and separating and removing the same volume of the culture medium from the tissues or the cells, and the taxane-type diterpene is recovered from at least one selected from the resulting tissues and/or cells, the culture medium recovered during the culture and obtained at the end of the culture. The method according to claim 1, wherein a fresh medium is added continuously or intermittently in such a way that the specific renewing ratio defined by the dimensionless number F = V I /V/µ (wherein, V is the total volume of the culture solution in a culture tank, V I is the feed speed of the fresh medium, and µ is the specific growth rate of the tissues or the cells) is in the range of 0.1 to 10, and the taxane-type diterpene is recovered from the culture medium and the tissues or the cells contained in the culture medium which is continuously or intermittently taken out from the tank and/or from the culture medium containing no tissue and cell which is continuously or intermittently taken out from the tank. The method according to claim 1, wherein the tissues or the cells of the plant which produce the taxane-type diterpene are cultured by the use of oxygenic gas containing 0.03 - 10 % of carbon dioxide to be introduced to the culture vessel.

Inventors: HARA YOSUHIRO, HIGASHI YOSUKE, MATSUBARA KOUICHI, OHNISHI NAOTO, SUGA CHUZO, TABATA HOMARE, YUKIMUNE YUKIHITO, HARA, YOSUHIRO, HIGASHI, YOSUKE, MATSUBARA, KOUICHI, OHNISHI, NAOTO, SUGA, CHUZO, TABATA, HOMARE, YUKIMUNE, YUKIHITO, Hara, Yosuhiro, c/o Mitsui Chemicals, Inc., Higashi, Yosuke, c/o Mitsui Chemicals, Inc., Matsubara, Kouichi, c/o Mitsui Chemicals, Inc., Ohnishi, Naoto, c/o Mitsui Chemicals, Inc., Suga, Chuzo, c/o Mitsui Chemicals, Inc., Tabata, Homare, c/o Mitsui Chemicals, Inc., Yukimune, Yukihito, c/o Mitsui Chemicals, Inc.
Applicants: MITSUI CHEMICALS INC, MITSUI CHEMICALS, INC.
Application year: 1994
Main classification: C12P17
IPC codes: C12N5, C12P17